WO2020115510A1 - Compositions d'hydrogel oculaires - Google Patents

Compositions d'hydrogel oculaires Download PDF

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Publication number
WO2020115510A1
WO2020115510A1 PCT/GB2019/053479 GB2019053479W WO2020115510A1 WO 2020115510 A1 WO2020115510 A1 WO 2020115510A1 GB 2019053479 W GB2019053479 W GB 2019053479W WO 2020115510 A1 WO2020115510 A1 WO 2020115510A1
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Prior art keywords
shear
thinning
hydrogel composition
ocular
decorin
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PCT/GB2019/053479
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English (en)
Inventor
Liam Grover
Anthony Metcalfe
Richard Williams
Richard Moakes
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The University Of Birmingham
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Priority to EP19821193.0A priority Critical patent/EP3890702A1/fr
Priority to JP2021532375A priority patent/JP2022517722A/ja
Priority to KR1020217020673A priority patent/KR20210113200A/ko
Priority to US17/311,561 priority patent/US20220023206A1/en
Priority to CN201980091280.2A priority patent/CN113473966A/zh
Publication of WO2020115510A1 publication Critical patent/WO2020115510A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A61K9/0051—Ocular inserts, ocular implants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4725—Proteoglycans, e.g. aggreccan

Definitions

  • the present invention relates to hydrogel compositions that are useful for therapeutic application in the eye.
  • the present invention further relates to method for preparing these hydrogel compositions and their use for therapeutic applications, especially in the inhibition of ocular scarring.
  • Corneal opacity is a leading cause of sight impairment worldwide with an estimated 27.9 million people globally being bilaterally or unilaterally affected 111 . Such opacity is typically derived from alteration of the complex, optically clear, corneal tissue structure, vital for refraction of light onto the retina, and subsequent neuro-visual processing. Commonly, corneal scarring results from ocular infections from a range of pathogens including bacteria, parasites, fungi, viruses and protozoa. In the developed world, devastating corneal infections are most commonly associated with prolonged contact lens wear and/or poor lens hygiene 12-41 ; with Pseudomonas aeruginosa being a prominent causative organism. In cases of gram-negative infections, e.g.
  • the structural integrity of the cornea becomes compromised through multiple virulence factors, whereby the microbes invade epithelial cells, resulting in activation of numerous inflammatory pathways.
  • Subsequent production of cytokines from epithelial, stromal and intraepithelial inflammatory cells, neovascularization, cellular alterations and degradative stromal processes[5] lead to dysregulated tissue remodeling and disruption of the intricately arranged collagen fibrils[6] leading to the loss of optical transparency, impairment of light refraction and loss of sight.
  • an orchestrated wound healing response involving corneal epithelium, stroma and nerves, lacrimal glands and tear film occurs to restore corneal structure and function and maintain the ocular integrity 171 .
  • the epithelium starts to regenerate in response to stem cell proliferation from the limbal niche almost immediately after the epithelium is injured 181 and keratocytes (transparent cells that function to maintain collagen and ECM turnover) proximal to the wounding site undergo apoptosis (induced by cytokines released from damaged epithelial cells).
  • Proliferation and migration of residual keratocytes peripheral to the injury-site can be detected 12 to 24 hours after injury 191 .
  • the keratocytic response includes production of proteoglycans and synthesis of collagen fibers. These fibers are phenotypically larger than the naive fiber and, due to the water-retention capacity of the proteoglycans, do not assume an ordered regular architecture leading to corneal opacity.
  • the movement of bone-marrow derived precursor cells and circulatory mediators from the from the limbal region activate, transform and differentiate a subset of keratocytes to cell types with fibroblast and myofibroblast characteristics via TQEb and PDGF TQRb released from epithelial cells accesses stromal cells through the damaged Bowman’s layer to initiate myofibroblast differentiation 112 ⁇ 131 .
  • Myofibroblasts release cytokines that further attract inflammatory cells and ECM deposition (e.g. collagen, fibronectin) to facilitate the fibroblast migration as part of the stromal remodeling phase 1141 . .
  • Reparative processes lead to improved collagen fiber orientation, contraction of proteoglycan, apoptosis of the stromal myofibroblasts and repopulation of keratocytes enable structural and functional recovery, but with residual stromal opacity. If this is present in the visual axis, sight is impaired. If the corneal epithelial barrier is not restored, stromal metabolism becomes dysregulated leading to keratolysis, degradation of corneal tissue, further disorganization of corneal fibril arrangements, and eventual corneal perforation. This is partly due to the sustained release of TQRb leading to persistent myofibroblasts preventing stromal re-population of keratocytes [15 ⁇ 16] . Unlike other tissues (e.g. skin), where a persistent ulcer or scar might be tolerated, in the cornea this can have devastating functional effects of permanent corneal scarring with visual disability or loss of eye.
  • Surgical interventions to treat unresponsive and large corneal defects include either application of amniotic membrane as a biologically active bandage releasing anti inflammatory and anti-fibrotic factors to enhance re-epithelialization and wound healing during acute injury 112-141 , or in established cases of visually significant central corneal scars, excision of the scarred tissue and replacement with donor cornea. Reproducibility and repeatability of the clinical outcomes of amnion grafting and corneal transplantation are fraught with risks of failure and
  • fibrosis is driven by raised levels of TQRb-1 activity and so it may be possible to prevent fibrosis using a TQRb antagonist.
  • Decorin is a naturally occurring pleiotropic anti-fibrotic small leucine-rich proteoglycan that is naturally present at high levels bound to collagen in the corneal stroma 22 and which, when released, tightly regulates TQRb activity by binding the growth factor and sequestering it within the ECM [191 .
  • Decorin regulates cell proliferation, survival and differentiation by modulating numerous growth TGF-b as well as directly interfering with collagen
  • Decorin is responsible for regulating collagen fibril spacing and ECM to enable corneal transparency and has previously been shown to inhibit scar formation and neovascularization in the cornea [35] .
  • Mutations in decorin are associated with corneal opacities and visual abnormalities associated with congenital stromal dystrophy [401 .
  • Hyperactivity of TQRb in corneal fibrosis may overcome the ability of endogenous decorin to maintain homeostasis and there is good evidence that over-expressing decorin in other tissues is able to reduce levels of fibrosis i
  • a shear-thinning ocular hydrogel composition suitable for application to the eye, the composition comprising:
  • hydrogel composition has a pH within the range of 3 to 8 and the viscosity of the hydrogel composition reduces when the hydrogel is exposed to shear
  • composition further comprises decorin.
  • an ocular hydrogel composition suitable for application to the eye, wherein the ocular hydrogel composition comprises, consist essentially of, or consists of, a shear-thinning hydrogel composition comprising decorin as defined herein.
  • the present invention provides a method of making a shear thinning ocular hydrogel composition as defined herein, the method comprising the steps of: a) dissolving a microgel-forming polymer in an aqueous vehicle to form a polymer solution;
  • step (b) mixing the microgel-forming polymer solution formed in step (a) with an aqueous solution of a monovalent or polyvalent metal ion salt at a temperature above the gelling temperature of the microgel particle-forming polymer;
  • step b) cooling the resultant mixture from step b) to a temperature below the gelling temperature of the microgel particle-forming polymer
  • the decorin is added to the mixture in the form of an aqueous decorin solution.
  • the present invention provides a method of making a shear thinning ocular hydrogel composition as defined herein, the method comprising the steps of: a) dissolving a microgel-forming polymer in an aqueous vehicle comprising 0.5 to 100mM of a monovalent and/or polyvalent metal ion salt as a cross-linking agent to form a polymer solution comprising 0.1 to 5.0wt.% (e.g. 0.1 to 3.5wt.%, or 0.1 to 2.5wt%) of the microgel particle-forming polymer;
  • step b) cooling the resultant mixture from step a) under shear mixing to a temperature below the gelling temperature of the microgel particle-forming polymer;
  • step b) at a point wherein the mixture from step a) is at a temperature above the gelling temperature of the microgel particle-forming polymer.
  • the decorin is added to the mixture in the form of an aqueous decorin solution.
  • the present invention provides a shear-thinning ocular hydrogel composition obtainable by, obtained by, or directly obtained by, any of the preparatory methods defined herein.
  • the present invention provides a shear-thinning ocular hydrogel composition as defined herein for use in therapy.
  • the present invention provides a shear-thinning ocular hydrogel composition as defined herein for ocular administration.
  • the present invention provides a shear-thinning ocular hydrogel composition as defined herein for use in the inhibition of scarring.
  • the present invention provides a shear-thinning ocular hydrogel composition as defined herein for use in the treatment of microbial keratitis.
  • the invention provides an ocular composition in accordance with the invention for use as a medicament.
  • a medicament for administration to a surface of the eye.
  • composition in accordance with the invention is for use in the inhibition of scarring in the eye.
  • the compositions of the invention may be used as medicaments for inhibition of scarring associated with keratitis, such as microbial keratitis.
  • the compositions of the invention may be used in the treatment of microbial keratitis to inhibit scarring.
  • the ocular hydrogel compositions of the invention comprise the anti-fibrotic ECM molecule decorin.
  • An ocular hydrogel composition in accordance with any of the aspects described herein may suitably contain no other biologically-derived agents, in particular no other biologically-derived agents from human or other animal sources.
  • an ocular hydrogel composition in accordance with the invention may comprise a polysaccharide microgel-forming polymer, but no protein components capable of gel formation.
  • compositions of the invention may not comprise a protein other than decorin.
  • an ocular hydrogel composition of the invention may contain no ECM components other than decorin.
  • an ocular hydrogel composition of the invention may not comprise collagen or fibrin.
  • the current invention is based upon the inventors’ surprising finding that anti-scarring ocular hydrogel compositions comprising the anti-fibrotic agent decorin can be improved by the exclusion of further ECM components, and particularly from the absence of collagen and fibrin from such compositions. This is in direct contrast to the reports of the prior art, which have indicated that collagen, and optionally fibrin, play vital roles in the ability of these compositions to inhibit scarring.
  • TGF-b and other bound factors are more effectively sequestered so that they cannot contribute to fibrosis, inflammation, or angiogenesis.
  • the sequestered factors are then removed from the eye surface as the fluid hydrogel is slowly blinked away.
  • an ocular hydrogel composition comprising decorin, but lacking collagen or fibrin (or indeed any other ECM), is highly effective in inhibiting scarring of the cornea, such as that associated with microbial keratitis.
  • additional ECM components such as collagen and/or fibrin
  • compositions of the invention that comprise decorin, but do not contain collagen or fibrin, are able to effectively inhibit scarring associated with microbial keratitis. Furthermore, the lack of ECM constituents able to interact with and “present” decorin, rather than hindering therapeutic effectiveness, actually confers advantages on the compositions of the invention.
  • ECM components including collagen and fibrin, incorporate motifs that allow them to bind to other biologically active molecules. It is this property that underpins prior suggestions regarding the use of collagen within compositions in order to present decorin in its native, and therefore more biologically active, context.
  • the favoured microgel particle forming polymers of the present invention such as gellan, lack motifs of this sort. Accordingly, it will be recognised they are not able to function in the manner ascribed to collagen and/or fibrin in the prior art, by binding to decorin and maintaining this agent in the conformation in which it is found in vivo.
  • Binding motifs on ECM components also have important roles in binding to cellular or soluble biological effector molecules. Such molecules provide signals or other biological cues to cells within the host, and the disruption of their signalling may influence the host’s response. While some of the alterations of the biological pathways (such as the binding and inhibition of fibrotic growth factors by decorin) have a beneficial therapeutic effect, this is not always the case. Other biological factors can have adverse impacts, causing sensitisation or inflammation at sites where they are provided. By excluding collagen and/or fibrin (or indeed ECM components other than decorin), the ocular hydrogel compositions of the invention are not subject to such undesirable biological effects.
  • ECM components such as collagen and/or fibrin
  • biologically- derived agents biomaterials that are typically obtained by extraction from naturally occurring sources. These sources, which may be human or other animal, provide naturally occurring proteins which have undergone“correct” processing to their biologically relevant forms (a result that is difficult to achieve by recombinant approaches).
  • biologically- derived agents can be subject to significant variation in respect of the sources from which they are derived. These can be“inter-source” variations (e.g. differences between products obtained from different individuals) or“intra-source” variations (e.g. differences in products obtained from the same individual at different times). Intra-source variations may be exacerbated by factors such as health status or medication.
  • ocular hydrogel compositions of the invention as compared to those of the prior art, is that, by utilising gel materials (such as gellan) that are not obtained by extraction from human or animal sources they avoid such variation in their properties.
  • compositions of the invention allow for the retention of biologically active decorin at the surface of the cornea, thereby improving the availability of the decorin, and hence its capacity to inhibit scar formation. This is achieved without the need for collagen associated or complexed with the decorin. Instead, these benefits arise as a result of the material properties of the shear-thinning ocular hydrogel compositions.
  • the properties of the fluid gels employed in the compositions of the invention are such that the decorin is held within the ocular hydrogel composition at the eye’s surface.
  • the hydrogel provides a protective layer over damaged sites, such as infections associated with microbial keratitis, contributing to the develonce of a therapeutic healing milieu.
  • the shear-thinning ocular hydrogel compositions of the invention do not only contribute to the inhibition of scarring through protection of the damaged area.
  • the inventors have found that the material properties of the shear-thinning ocular hydrogel are such that a semi-solid to liquid transition occurs when they are exposed to shear forces consistent with those generated by blinking (i.e. the forces that result from the interaction of the compositions with a recipient’s eyelids during blinking). This liquefaction is believed to cause pulsatile release of anti-fibrotic decorin with each blink. After blinking the composition returns to its semi-solid form, effectively storing the remaining decorin in a depot. The regular and controlled release of decorin in this manner establishes highly favourable conditions in which scar-free healing of the ocular surface is able to occur.
  • shear-thinning hydrogels used in the ocular compositions of the present invention rely upon an ability to undergo repeated transitions between liquid and semi-solid transitions in order to achieve this effect.
  • Such fluid gels may be termed “self-healing”.
  • the gelation mechanism for fibrin is irreversible. Accordingly, the incorporation of fibrin in embodiments of the gels described in the prior art results in compositions that once“broken” are unable to“heal”.
  • Collagen another ECM component disclosed as a“carrier” for decorin in the prior
  • a“carrier” for decorin in the prior is also unsuitable for the formation of fluid gels.
  • the mixing required for formation of such gels causes collagen to fail to gel appropriately during the manufacturing process.
  • prior art gels based on collagen and/or fibrin lack the ability to generate regular“pulses” of decorin during their residency in the eye, and so are not able to confer the benefits provided by the ocular hydrogel compositions of the invention.
  • the ocular hydrogel compositions of the present invention also offer benefits in terms of the way in which they are manufactured that are not provided for by the compositions of the prior art.
  • the shear-thinning ocular hydrogel compositions of the invention offer benefits on the basis of their reproducibility of manufacture, their ease of manufacture, and the supply chain involved in manufacturing and distributing the product.
  • Ensuing continuity and consistency of supply of biologically-sourced materials also introduces difficulties in the supply of manufacturing materials to be used, and the distribution of the compositions produced.
  • Enzymes and their substrates can be sensitive to the way in which they are handled (since variations in temperature, or the like, may significantly impact upon activity), and biological gels of this sort typically require refrigerated (or frozen) storage after manufacture.
  • Use of biological material, such as collagen or fibrin is also associated with the risk of unwanted contaminants, such as infections agents, being introduced into the products.
  • Figure 1 Processing and intrinsic material properties of the gellan based fluid hydrogel eye drop, (a) Schematic showing the production of the fluid gel: where the initial sol is continuously processed under shear whilst being cooled to form“ribbon-like” gelled entities shown using (i) transmission microscopy and (ii) scanning electron microscopy (b) Time dependent viscosity profiles obtained for the gellan eye drop, highlighting a degree of thixotropy (c) The fluid gel being dispensed from the eye dropper packaging (gel has been stained blue so as to be visible in the photograph) (d) Small deformation rheology data obtained at a single frequency (1 Hz, 0.5% strain) as a function of time.
  • Figure 4 Corneal re-epithelialization.
  • DecFG (scale bar 100 mm)
  • FIG. 5 Extracellular matrix levels in the cornea. Representative images of immunohistochemical staining with accompanying plots quantifying the IR for: (a) aSMA + (green to stain myofibroblasts), (b) IR fibronectin + (green to stain fibronectin in the ECM), and (c) laminin + (red to stain laminin in the ECM), in each case DAPI + was used to stain the cell nuclei (blue).
  • Figure 6 In vivo experimental design. Experimental design for the in vivo Pseudomonas keratitis study in which the fluid gel eye drops with and without hrDecorin were compared to Gentamicin and Prednisolone eye drops alone.
  • Figure 7 Storage modulus (G’) representing the elastic structure within the gellan microgel suspensions, as a function of initial gellan polymer concentration; determined using amplitude sweeps (a) strain sweeps obtained at 1 Hz (20 °C) for varying polymer concentrations prepared at a processing rate of 500 rmm. (b) strain sweeps obtained at 1 Hz (20 °C) for varying polymer concentrations at a processing rate of 1000 rmm.
  • FIG. 8 Comparison of storage moduli as a function of polymer concentration and processing speeds. G’ obtained within the linear viscoelastic region (LVR) of the amplitude sweeps shown in Fig.7
  • Figure 9 Comparison in storage moduli for commercially available eye drops/ointments for the treatment of dry eye. Data obtained from amplitude sweeps undertaken using the same method as described for gellan suspensions. Again, values were obtained within the LVR. Dotted line represents G’ for the optimised gellan formulation.
  • Figure 10 Flow profiles representing the ease of application for the gellan microgel suspensions, as a function of initial gellan polymer concentration (a) Viscosity sweeps obtained at 20 °C between 0.1 and 600 s -1 for varying polymer concentrations prepared at a processing rate of 500 rmm. (b) Viscosity sweeps obtained at 20 °C between 0.1 and 600 s 1 for varying polymer concentrations prepared at a processing rate of 1000 rmm.
  • Figure 11 Comparison of the microgel suspension viscosity at 1 s -1 a function of polymer concentration and processing speeds. Instantaneous viscosity was obtained by measuring the value at 1 s _1 using the sweeps shown in Fig.10.
  • Figure 12 Comparison in viscosities at 1 s ⁇ 1 for commercially available eye drops/ointments for the treatment of dry eye. Data obtained from flow profiles undertaken using the same method as described for gellan suspensions. Dotted line represents the viscosity of the optimised gellan formulation.
  • Figure 13 Storage modulus (G’) representing the elastic structure within the gellan microgel suspensions, as a function of cross-linker added; determined using amplitude sweeps (a) strain sweeps obtained at 1 Hz (20 °C) for varying cross-linker concentrations for 0.9% (w/v) systems (b) strain sweeps obtained at 1 Hz (20 °C) for varying cross-linker concentrations for 1.8% (w/v) polymer concentrations.
  • Figure 14 Comparison of storage moduli as a function the cross-linker and polymer concentrations. G’ obtained within the linear viscoelastic region (LVR) of the amplitude sweeps shown in Fig.7.
  • Figure 15 Flow profiles representing the ease of application for the gellan microgel suspensions, as a function of the cross-linker concentration.
  • Figure 16 Comparison of the microgel suspension viscosities at 1 s -1 as a function of the polymer and cross-linker concentrations. Instantaneous viscosity was obtained by measuring the value at 1 s -1 using the sweeps shown in Fig.3.
  • Figure 17 Storage modulus (G’) representing the elastic structure within the gellan microgel suspensions, as a function of cooling rate applied during processing; determined using amplitude sweeps, (a) strain sweeps obtained at 1 Hz (20 °C) for varying cooling rates for 0.9% (w/v) systems prepared at a processing rate of 1000 rmm. (b) strain sweeps obtained at 1 Hz (20 °C) for varying cooling rates for1.8% (w/v) polymer concentrations at a processing rate of 1000 rmm.
  • G Storage modulus
  • Figure 18 Comparison of storage moduli as a function of cooling rate and polymer concentration. G’ obtained within the linear viscoelastic region (LVR) of the amplitude sweeps shown in Fig.7.
  • Figure 19 Flow profiles representing the ease of application for the gellan microgel suspensions, as a function of the cooling rate applied during processing, (a) Viscosity sweeps obtained at 20 °C between 0.1 and 600 s -1 for 0.9% (w/v) gellan systems prepared at various cooling rates (b) Viscosity sweeps obtained at 20 °C between 0.1 and 600 s for 1.8% (w/v) gellan systems prepared at various cooling rates.
  • Figure 20 Comparison of the microgel suspension viscosities at 1 s -1 as a function of polymer concentration and cooling rate applied during processing. Instantaneous viscosity was obtained by measuring the value at 1 s -1 using the sweeps shown in Fig.9.
  • Figure 21 Storage modulus (G’) representing the elastic structure within the gellan microgel suspensions, as a function of mechanical shear applied during processing; determined using amplitude sweeps, (a) strain sweeps obtained at 1 Hz (20 °C) for varying processing speeds for 0.9% (w/v) systems (b) strain sweeps obtained at 1 Hz (20 °C) for varying processing speeds for 1.8% (w/v) polymer concentrations.
  • G Storage modulus
  • Figure 22 Comparison of storage moduli as a function of processing speeds and polymer concentration. G’ obtained within the linear viscoelastic region (LVR) of the amplitude sweeps shown in Fig.7.
  • Figure 23 Flow profiles representing the ease of application for the gellan microgel suspensions, as a function of the mechanical shear applied during processing, (a) Viscosity sweeps obtained at 20 °C between 0.1 and 600 s _1 for 0.9% (w/v) gellan systems prepared at various processing speeds (b) Viscosity sweeps obtained at 20 °C between 0.1 and 600 s- for 1.8% (w/v) gellan systems prepared at various processing speeds.
  • Figure 24 Comparison of the microgel suspension viscosities at 1 s ⁇ 1 as a function of polymer concentration and processing speeds during gelation. Instantaneous viscosity was obtained by measuring the value at 1 s -1 using the sweeps shown in Fig.9.
  • Figure 25 shear-thinning hydrogel compositions in accordance with the invention reduce expression in cultured fibroblasts of markers associated with scarring.
  • TGF-b to cultured human dermal fibroblasts increases expression of a- smooth muscle actin, a marker of myofibroblasts associated with scarring.
  • Graphs show the impact of treatment with experimental hydrogel compositions on this expression.
  • Hydrogel ocular compositions of the invention are able to reduce expression of a-sma, indicating an ability to inhibit scarring.
  • hydrogel is used herein to refer to a gel formed from a hydrophilic polymer dispersed within an aqueous vehicle.
  • aqueous vehicle is used herein to refer to water or water-based fluid (e.g. a buffer such as, for example, phosphate buffered saline or a physiological fluid such as, for example, serum).
  • a buffer such as, for example, phosphate buffered saline
  • physiological fluid such as, for example, serum
  • microgel is used herein to refer to a microscopic particle of gel formed from a network of microscopic filaments of polymer.
  • shear-thinning is used herein to define the hydrogel compositions of the present invention. This terminology is well understood in the art and refers to hydrogel compositions that have a viscosity that reduces when a shear force is applied to the hydrogel.
  • the shear-thinning hydrogel compositions of the invention possess a“resting” viscosity (in the absence of any applied shear force), and a lower viscosity when a shear force is applied. This property of hydrogel compositions enables them to flow and be administered to the body when a shear force is applied (for example, by applying a force to a tube or dispenser containing the hydrogel composition of the invention).
  • the viscosity of hydrogel composition increases.
  • the hydrogel compositions of the present invention will have a viscosity of below 1 Pa.s when subjected to a shear force to administer the hydrogel composition.
  • the hydrogel composition will be capable of flowing.
  • the resting viscosity will typically be above 1 Pa.s, for example greater than 2 Pa.s, greater than 3 Pa.s, or greater than 4 Pa.s.
  • references to “treating” or “treatment” include prophylaxis as well as the alleviation of established symptoms of a condition.“Treating” or “treatment” of a state, disorder or condition therefore includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (2) inhibiting the state, disorder or condition, i.e.
  • A“therapeutically effective amount” means the amount of a compound that, when administered to a mammal for treating a disease, is sufficient to effect such treatment for the disease.
  • the "therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the mammal to be treated. Further considerations regarding therapeutically effective amounts of ocular hydrogel compositions of the invention, or of decorin or other agents to be incorporated in such ocular hydrogel compositions, are considered in more detail elsewhere in the specification.
  • the present invention is directed to shear-thinning ocular hydrogel compositions, and, unless context requires otherwise, all references within the present disclosure to “hydrogels”, “compositions”, or“hydrogel compositions” should be taken as relating to “shear-thinning ocular hydrogel compositions”.
  • a shear-thinning ocular hydrogel composition comprising:
  • hydrogel composition has a pH within the range of 3 to 8 and the viscosity of the hydrogel composition reduces when the hydrogel is exposed to shear;
  • composition comprises decorin.
  • the ocular hydrogel compositions of the present invention are shear-thinning, meaning that the viscosity of the composition reduces when the hydrogel is exposed to shear. This property enables the hydrogels to reduce in viscosity and flow when a shear force is applied, thereby enabling them to be dispensed and administered, for example from an eye dropper to tube, by applying a shear force (e.g. by squeezing the sides of the eye dropper or tube). Once administered and the shear force applied to the hydrogel diminishes, the viscosity of the hydrogel increases to form a thicker gel capable of residing at the point of administration for a prolonged period.
  • a shear force e.g. by squeezing the sides of the eye dropper or tube
  • the hydrogel compositions of the present invention will have a viscosity of below 1 Pa.s when subjected to a shear force to administer the hydrogel composition. At viscosities below 1 Pa.s, the hydrogel composition will be capable of flowing. The resting viscosity will typically be above 1 Pa.s, for example greater than 2 Pa.s, greater than 3 Pa.s, or greater than 4 Pa.s.
  • the microgel particle-forming polymer may be any polymer that is capable of forming microgel particles in the aqueous vehicle.
  • the microgel particles formed by the microgel particle-forming polymer may have any suitable morphology (e.g. they may be linear filaments or regular or irregular shaped particles) and/or particle size.
  • the formation of microgel particles facilitates the desired shear thinning characteristics. Without wishing to be bound by any particular theory, it is postulated that, in the absence of shear or at low levels of shear, the microgel particles are bound together, substantially impeding the bulk flow of the hydrogel.
  • the shear-thinning hydrogel compositions of the present invention do not comprise collagen and/or fibrin.
  • the hydrogel composition comprises 0.1 to 5.0wt.% of the microgel particle forming polymer. In an embodiment, the hydrogel composition comprises 0.1 to 3.5wt.% of the microgel particle-forming polymer. In an embodiment, the hydrogel composition comprises 0.5 to 2.5 wt.% of the microgel particle-forming polymer. In an embodiment, the hydrogel composition comprises 0.8 to 1.8 wt.% of microgel particle-forming polymer. In a further embodiment, the hydrogel composition comprises 0.8 to 1.0 wt.% (e.g. 0.9 wt.%) of a microgel particle-forming polymer.
  • microgel particles are formed from one or more polysaccharide microgel particle-forming polymers.
  • microgel particles are not formed from decorin.
  • the microgel particle-forming polymer is one or more polysaccharide microgel particle-forming polymers.
  • the microgel particle-forming polymer is selected from one or more of the following groups: gellans, alginates, carrageenans, agarose.
  • the microgel particle-forming polymer is selected from one or more of the following groups: gellans, alginates or carrageenans.
  • the microgel particle-forming polymer is selected from gellan or alginate.
  • the microgel particle-forming polymer is gellan.
  • the microgel particle-forming polymer is an alginate.
  • the microgel particle-forming polymer is gelatin.
  • the ocular hydrogel compositions of the invention are transparent or translucent. In a particular embodiment, the ocular hydrogel composition is transparent.
  • the hydrogel composition is transparent or translucent and the microgel particle-forming polymer is selected from gellans, alginates and/or carrageenans. In a further embodiment, the hydrogel composition is transparent and the microgel particle forming polymer is selected from gellans, alginates and/or carrageenans. In a particular embodiment, the hydrogel composition is transparent and the microgel particle-forming polymer is gellan or alginate. In a further embodiment, the hydrogel composition is transparent and the microgel particle-forming polymer is gellan. [0067] Gellan (also referred to gellan gum) is a water-soluble anionic polysaccharide produced by the bacterium Sphingomonas elodea. If is commercially available in a low acyl form under the trade name Kelco gel (Kelco gel CG LA, Azelis, UK).
  • the hydrogel composition comprises 5 to 100 mM of a monovalent and/or polyvalent metal ion salt as a cross-linking agent.
  • the metal ion salt may be added to the composition as a component, but it may also be present in other components of the composition, e.g. components such as buffers (e.g. phosphate buffered saline) or any physiological fluids present in the composition, such as, for example, serum.
  • the hydrogel composition comprises 5 to 40 mM of a monovalent and/or polyvalent metal ion salt as a cross-linking agent.
  • the hydrogel composition comprises 5 to 30 mM of a monovalent and/or polyvalent metal ion salt as a cross-linking agent.
  • the hydrogel composition comprises 5 to 20 mM of a monovalent and/or polyvalent metal ion salt as a cross-linking agent.
  • the hydrogel composition comprises 5 to 15 mM of a monovalent and/or polyvalent metal ion salt as a cross-linking agent.
  • the hydrogel composition comprises 8 to 12 mM (e.g. 10 mM) of a monovalent and/or polyvalent metal ion salt as a cross-linking agent.
  • the microgel particle-forming polymer is gellan and the composition comprises 0.5 to 40 mM, 5 to 15 mM, 8 to 12 mM or 10 mM of a monovalent metal ion salt (e.g. NaCI) as a cross-linking agent.
  • a monovalent metal ion salt e.g. NaCI
  • the microgel particle-forming polymer is alginate and the composition comprises 0.5 to 40 mM, 5 to 15 mM, 8 to 12 mM or 10 mM of a polyvalent metal ion salt (e.g. a Ca 2+ salt) as a cross-linking agent.
  • a polyvalent metal ion salt e.g. a Ca 2+ salt
  • the hydrogel composition has a pH within the range of 6 to 8. In an embodiment, the hydrogel composition has a pH within the range of 6.5 to 8. In a further embodiment, the hydrogel composition has a pH within the range of 7 to 7.5 (e.g. pH 7.4).
  • the hydrogel composition of the present invention has a resting viscosity (i.e. a viscosity at zero shear) of 1 Pa.s or greater (e.g. 1 Pa.s to 200 Pa.s or 1 Pa.s to 100 Pa.s). More suitably, the resting viscosity will be 2 Pa.s or greater (e.g. 2 Pa.s to 200 Pa.s or 2 Pa.s to 100 Pa.s), 3 Pa.s or greater (e.g. 3 Pa.s to 200 Pa.s or 3 Pa.s to 100 Pa.s), 4 Pa.s or greater (e.g. 4 Pa.s to 200 Pa.s or 4 Pa.s to 100 Pa.s), or 5 Pa.s or greater (e.g. 5 Pa.s to 200 Pa.s or 5 Pa.s to 100 Pa.s).
  • a resting viscosity i.e. a viscosity at zero shear
  • 1 Pa.s or greater e.g. 1 Pa.s to 200 Pa.s or 1 Pa.
  • the viscosity reduces when the hydrogel composition is subjected to a shear force.
  • the viscosity reduces to a value below the resting viscosity at which the gel can flow and be administered.
  • the viscosity will reduce to a value of less than 1 Pa.s when a shear force is applied.
  • the hydrogel composition has a resting viscosity of 1 Pa.s or greater (e.g. 1 Pa.s to 200 Pa.s or 1 Pa.s to 100 Pa.s) and when subject to a shear force, the viscosity reduces to below 1 Pa.s.
  • the hydrogel composition has a resting viscosity of 2 Pa.s or greater (e.g. 2 Pa.s to 200 Pa.s or 2 Pa.s to 100 Pa.s) and when subject to a shear force, the viscosity reduces to below 2 Pa.s (for example, to below 1 Pa.s).
  • the hydrogel composition has a resting viscosity of 3 Pa.s or greater (e.g. 3 Pa.s to 200 Pa.s or 3 Pa.s to 100 Pa.s) and when subject to a shear force, the viscosity reduces to below 3 Pa.s (for example, to below 1 Pa.s).
  • the hydrogel composition has a resting viscosity of 4 Pa.s or greater (e.g. 4 Pa.s to 200 Pa.s or 4 Pa.s to 100 Pa.s) and when subject to a shear force, the viscosity reduces to below 4 Pa.s (for example, to below 1 Pa.s).
  • the hydrogel composition has a resting viscosity of 5 Pa.s or greater (e.g. 5 Pa.s to 200 Pa.s or 5 Pa.s to 100 Pa.s) and when subject to a shear force, the viscosity reduces to below 5 Pa.s (for example, to below 1 Pa.s).
  • viscosity values quoted herein are quoted at a normal ambient temperature of 20°C.
  • the viscosity of hydrogel compositions of the present invention can be determined using standard techniques well known in the art. For example, viscosity profiles can be obtained using an AR-G2 (TA Instruments, UK) rheometer equipped with sandblasted parallel plates (40 mm, 1 mm gap height) at 20 °C.
  • the hydrogel has an elastic modulus of 5 Pa to 40 Pa at zero shear.
  • the elastic modulus of the hydrogels of the present invention can be determined by techniques well known in the art.
  • shear-thinning ocular hydrogel composition comprises decorin and:
  • 0.1 to 5.0 wt.% e.g. 0.1 to 3.5wt.% or 0.1 to 2.5 wt.% of a microgel particle-forming polymer (e.g. gellan);
  • hydrogel composition has a pH of 3.5 to 8.
  • a monovalent metal ion salt e.g. NaCI
  • polyvalent metal ion salt e.g. Ca 2+
  • 0.1 to 5.0 wt.% e.g. 0.1 to 3.5 wt.% or 0.1 to 2.5wt.% of a microgel particle-forming polymer (e.g. gellan);
  • a monovalent metal ion salt e.g. NaCI
  • polyvalent metal ion salt e.g. Ca 2+
  • the hydrogel composition has a pH of 6 to 8.
  • a microgel particle-forming polymer e.g. gellan
  • a monovalent metal ion salt e.g. NaCI
  • polyvalent metal ion salt e.g. Ca 2+
  • the hydrogel composition has a pH of 6.5 to 7.5.
  • microgel particle-forming polymer e.g. gellan
  • a monovalent metal ion salt e.g. NaCI
  • polyvalent metal ion salt e.g. Ca 2+
  • the hydrogel composition has a pH of 3.5 to 8.
  • microgel particle-forming polymer e.g. gellan
  • a monovalent metal ion salt e.g. NaCI
  • polyvalent metal ion salt e.g. Ca 2+
  • the hydrogel composition has a pH of 6 to 8.
  • microgel particle-forming polymer e.g. gellan
  • a monovalent metal ion salt e.g. NaCI
  • polyvalent metal ion salt e.g. Ca 2+
  • the hydrogel composition has a pH of 6.5 to 7.5.
  • microgel particle-forming polymer e.g. gellan
  • a monovalent metal ion salt e.g. NaCI
  • polyvalent metal ion salt e.g. NaCI
  • the hydrogel composition has a pH of 3.5 to 8.
  • microgel particle-forming polymer e.g. gellan
  • hydrogel composition has a pH of 6 to 8.
  • a monovalent metal ion salt e.g. NaCI
  • polyvalent metal ion salt e.g. Ca 2+
  • the hydrogel composition has a pH of 6 to 8.
  • microgel particle-forming polymer e.g. gellan
  • a monovalent metal ion salt e.g. NaCI
  • polyvalent metal ion salt e.g. Ca 2+
  • the hydrogel composition has a pH of 6 to 8.
  • microgel particle-forming polymer e.g. gellan
  • the hydrogel composition has a pH of 6 to 8.
  • microgel particle-forming polymer e.g. gellan
  • a monovalent metal ion salt e.g. NaCI
  • polyvalent metal ion salt e.g. Ca 2+
  • the hydrogel composition has a pH of 6 to 8.
  • microgel particle-forming polymer e.g. gellan
  • a monovalent metal ion salt e.g. NaCI
  • polyvalent metal ion salt e.g. Ca 2+
  • the hydrogel composition has a pH of 6 to 8.
  • microgel particle-forming polymer e.g. gellan
  • a monovalent metal ion salt e.g. NaCI
  • polyvalent metal ion salt e.g. Ca 2+
  • the hydrogel composition has a pH of 6 to 8.
  • microgel particle-forming polymer e.g. gellan
  • the hydrogel composition has a pH of 6 to 8.
  • the shear-thinning ocular hydrogel compositions of the invention comprise the pharmaceutically active therapeutic agent decorin.
  • the hydrogel composition may comprise one or more further pharmacologically active agents. Any suitable pharmacologically active agent may be present.
  • the hydrogel composition may comprise one or more further pharmacologically active agents selected from the group consisting of: a further anti-fibrotic agent; an anti-infective agent; and an anti-inflammatory agent.
  • the ocular hydrogel composition comprises decorin in an amount of from 0.1 to 1.0 mg/ml; 0.1 to 0.5 mg/ml; 0.1 to 0.4 mg/ml; or 0.2 to 0.3 mg/ml.
  • the ocular hydrogel composition comprises decorin in an amount of from 0.1 to 1.0 mg/ml; 0.1 to 0.5 mg/ml; 0.1 to 0.4 mg/ml; or 0.2 to 0.3 mg/ml, in any one of the hydrogel compositions defined in paragraphs (1) to (14) above.
  • the ocular hydrogel composition may comprise any suitable amount of a further pharmacologically active agent.
  • the hydrogel composition may comprise 0.01 to 50 wt.% of a further pharmacologically active agent.
  • composition of the invention comprising an anti-infective agent, such as the antibiotic gentamicin
  • an anti-infective agent such as the antibiotic gentamicin
  • this may be present in an amount of from 1 to 5 mg/ml.
  • an anti- infective agent such as gentamicin
  • an anti-infective agent such as gentamicin
  • An anti-infective agent, such as gentamicin may be present in an amount of from 2 to 4 mg/ml, or from 2.5 to 3.5 mg/ml.
  • composition of the invention comprising an anti-inflammatory agent, such as the steroid prednisolone
  • an anti-inflammatory agent such as prednisolone
  • an anti-inflammatory agent such as prednisolone may be present in an amount of from 1.25 to 170 mg/ml, for example from 1.25 to 50 mg/ml, or from 1.25 to 10 mg/ml.
  • the present invention further provides a method of making a shear-thinning ocular hydrogel composition as defined herein, the method comprising the steps of:
  • step (b) mixing the microgel-forming polymer solution formed in step (a) with an aqueous solution of a monovalent or polyvalent metal ion salt at a temperature above the gelling temperature of the microgel particle-forming polymer;
  • step b) cooling the resultant mixture from step b) to a temperature below the gelling temperature of the microgel particle-forming polymer
  • step b) i) during step b); or ii) during step c) at a point wherein the mixture from step b) is at a temperature above the gelling temperature of the microgel particle forming polymer.
  • step a) is performed by heating the microgel particle-forming polymer and aqueous vehicle to a temperature above the gelling temperature for the microgel particle forming polymer.
  • the gellan / aqueous vehicle mixture may be heated to 60 to 90°C (e.g. 70 °C) in order to dissolve the gellan polymer.
  • the amount of polymer dissolved will depend on the amount of polymer required in the hydrogel composition (i.e. it will be within the limits defined hereinbefore for the hydrogel composition).
  • step b) the solution formed in step a) is suitably maintained at a temperature above the gelation temperature for the microgel particle-forming polymer and is mixed with an aqueous solution of a monovalent or polyvalent metal ion salt.
  • the solution from step a) is continuously agitated before, during and/or after the addition of the solution of the monovalent or polyvalent metal ion salt.
  • the mixture may be mixed at a rate of 50 to 2000 revolutions per minute (rmm) to ensure thorough mixing.
  • a mixing rate of 300 to 900 rmm or 500 to 800 rmm may be used.
  • the mixing rate and mixing apparatus can be varied to provide a desired level of shear / agitation.
  • the gellan / aqueous vehicle solution from step a) may be cooled to a temperature of, for example, 35 to 50 °C (e.g. 40 °C) prior to mixture with a monovalent cation solution.
  • the amount of monovalent or polyvalent metal ion salt solution added will depend on the amount of metal ion salt required in the final hydrogel composition (i.e. it will be within the limits defined hereinbefore for the hydrogel composition).
  • step c) the mixture from step b) is cooled to a temperature below the gelation temperature for the microgel particle-forming polymer such that microgel particles form in the hydrogel composition.
  • the mixture from step b) is cooled gradually with constant mixing.
  • the mixture from step b) is cooled at a constant cooling rate with continuous agitation/shear applied. The cooling under agitation/shear may continue until the mixture reaches ambient temperature (e.g. 20 °C), at which point the final hydrogel composition may be collected and stored, for example under refrigeration conditions.
  • the cooling rate used in step c) and the amount of shear/agitation applied can be varied.
  • a cooling rate of 0.2 to 4°C/min, 0.5 to 3°C/min, 0.5 to 2°C/min, 0.5 to 1.5°C/min, or 1 °C/min may be used.
  • the amount of shear applied may be, for example, 50 to 2000 rmm, 300 to 900 rmm, or 400 to 500 (e.g.450) rmm. Any suitable equimment may be used to provide the required agitation / shear.
  • a rotational rheometer AR-G2, TA Instruments, UK
  • cup and vane geometry cup: 35 mm diameter, vane: 28 mm diameter
  • Decorin may be added to the mixture in step b) or step c) of the method.
  • the decorin is added during step c) at a point where the mixture is above the gelling temperature for the microgel particle-forming polymer.
  • the mixture from step b) is cooled to a temperature above the gelling temperature for the microgel particle-forming polymer, the decorin is added and thoroughly mixed into the mixture, and the mixture is then further cooled to a temperature below the gelling temperature for the microgel particle forming polymer.
  • step b) is added to the mixture in either step b) or step c) in the form of an aqueous decorin solution.
  • a further pharmacologically active agent may be added during step b) or step c) (at a temperature above the gelling temperature of the microgel particle-forming polymer).
  • the present invention provides a method of making a shear thinning ocular hydrogel composition as defined herein, the method comprising the steps of: a) dissolving a microgel-forming polymer in an aqueous vehicle comprising 0.5 to 100mM of a monovalent and/or polyvalent metal ion salt as a cross-linking agent to form a polymer solution comprising 0.1 to 5.0 wt.% (e.g. 0.1 to 3.5 wt.% or 0.1 to 2.5wt%) of the microgel particle-forming polymer;
  • step b) cooling the resultant mixture from step a) under shear mixing to a temperature below the gelling temperature of the microgel particle-forming polymer;
  • step b) at a point wherein the mixture from step a) is at a temperature above the gelling temperature of the microgel particle-forming polymer.
  • the process is the same as the previous process defined above except that the microgel-particle forming polymer is dissolved directly in an aqueous vehicle comprising 0.5 to 100mM of a monovalent and/or polyvalent metal ion salt as a cross-linking agent.
  • aqueous vehicle comprising 0.5 to 100mM of a monovalent and/or polyvalent metal ion salt as a cross-linking agent.
  • Decorin may be added to the mixture in step a) or step b) of the method.
  • the decorin is added during step b) at a point where the mixture is above the gelling temperature for the microgel particle-forming polymer.
  • the mixture from step a) is cooled to a temperature above the gelling temperature for the microgel particle-forming polymer, the decorin is added and thoroughly mixed into the mixture, and the mixture is then further cooled to a temperature below the gelling temperature for the microgel particle forming polymer.
  • step a) or step b) Suitably decorin is added to the mixture in either step a) or step b) in the form of an aqueous decorin solution.
  • a further pharmacologically active agent may be added during step a) or step b) (at a temperature above the gelling temperature of the microgel particle-forming polymer).
  • the present invention provides a shear-thinning gel composition obtainable by, obtained by, or directly obtained by, any of the preparatory methods defined herein.
  • compositions of the invention and methods of treatment using the compositions of the invention
  • compositions of the invention for use as a medicament.
  • Compositions of the invention are suitable for medical use in the inhibition of scarring (as set out in a further aspect of the invention); as well as the prevention and/or treatment of infection; and the prevention and/or treatment of inflammation.
  • Compositions to be employed in such medical uses may comprise, as required, an active agent selected from the group consisting of: an anti-fibrotic agent; an anti-infective agent; and an anti inflammatory agent.
  • compositions of the invention are also suitable for use in methods of medical treatment.
  • compositions of the invention may be used in methods selected from the group consisting of: methods for the inhibition of scarring; methods for the prevention and/or treatment of infection; and methods for the prevention and/or treatment of inflammation.
  • a composition of the invention may be administered, as required, to a subject in need of inhibition of scarring; a subject in need of prevention and/or treatment of infection; or a subject in need of prevention and/or treatment of inflammation.
  • compositions of the invention may be used in methods for the inhibition of scarring in a subject that has microbial keratitis. Such use may also prevent and/or treat infection causing the microbial keratitis. Such use may also prevent and/or treat inflammation associated with microbial keratitis.
  • compositions to be employed in such methods of treatment may comprise, as required, an active agent selected from the group consisting of: a further anti- fibrotic agent (other than decorin); an anti-infective agent; and an anti-inflammatory agent.
  • an active agent selected from the group consisting of: a further anti- fibrotic agent (other than decorin); an anti-infective agent; and an anti-inflammatory agent.
  • scarring results in deleterious effects in many clinical contexts.
  • scarring of the eye may be associated with loss of sight, and risk of blindness.
  • an ocular composition of the invention for use in the inhibition of scarring may comprise gellan.
  • ocular compositions of the invention comprising gellan and decorin offer surprising benefits in the inhibition of scarring, as compared to prior art compositions.
  • the ocular hydrogel compositions of the invention that incorporate shear-thinning gellan hydrogels offer notable benefits as compared to those compositions of the prior art that employ ECM materials, such as collagen and/or fibrin.
  • Scarring in the eye includes scarring of the cornea, scarring of the retina, scarring of the ocular surface, and scarring in and around the optic nerve. Whilst the compositions of the invention are suitable for topical use, it will be appreciated that agents administered topically may have an effect on the internal anatomy. Thus, compositions administered to the surface of the eye may be effective in inhibiting intraocular scarring. Suitably scarring to be inhibited using the compositions or methods of the invention may include: scarring associated with infection, such as microbial keratitis; scarring associated with accidental injuries; and scarring associated with surgical injuries.
  • the ocular hydrogel compositions of the invention have particular utility in inhibiting scarring associated with keratitis. Keratitis may arise as a result of infection, for example microbial infection, viral infection, parasitic infection, or fungal infection.
  • the compositions and methods of the invention have shown particular utility in the inhibition of scarring associated with microbial keratitis.
  • Keratitis may also arise as a result of injury, or of disorders including autoimmune diseases such as rheumatoid arthritis or Sjogren’s syndrome.
  • the compositions and methods of the invention may also be used in inhibiting scarring associated with keratitis occurring as a result of these causes.
  • compositions of the invention may comprise a further pharmacologically active agent, such as, a further anti-fibrotic agent; an anti-infective agent; an anti inflammatory agent
  • Scarring in the eye that may be inhibited by the medical use of compositions of the invention may also include scarring associated with surgery, such as surgery for the treatment of glaucoma (for example by the insertion of stents); and surgical procedures such as LASIK or LASEK surgery, and scarring associated with accidental injuries.
  • scarring in the eye may be indicated by an increase in corneal opacity.
  • Such an increase in corneal opacity may be demonstrated by an increase in the area of the cornea that is opaque.
  • inhibition of scarring may be indicated by a reduction in corneal opacity as compared to a suitable control.
  • Such a decrease in corneal opacity may be demonstrated by a decrease in the area of the cornea that is opaque.
  • compositions of the invention comprising the anti-fibrotic agent decorin, to reduce corneal opacity, and to maintain such a reduction over time, is demonstrated in the data set out in the Examples.
  • scarring of the eye may be indicated by an increase in the presence of myofibroblasts.
  • inhibition of scarring may be indicated by a reduction in myofibroblast numbers as compared to a suitable control.
  • Myofibroblasts develop at the site of injuries and are associated with progression of the scarring response. They can be characterised by their expression of a-smooth muscle actin (a-sma). Myofibroblasts can have a number of adverse effects on scar formation, including causing contractions within the healed area. An increase in myofibroblasts associated with scarring may be demonstrated by an increase in a-smooth muscle actin expression. A reduction in myofibroblast numbers of this sort may be demonstrated by a decrease in a-smooth muscle actin expression.
  • the compositions of the invention are able to inhibit a-sma expression as assessed in vitro and in vivo, thus demonstrating their ability to inhibit scarring
  • compositions of the invention are able to inhibit myofibroblast differentiation in vivo, and are also able to maintain this reduced differentiation over time in an experimental model of microbial keratitis.
  • Myofibroblast differentiation may be increased in response to the action of TGF-bi, a fibrotic growth factor that causes induction of a-sma expression.
  • TGF-bi a fibrotic growth factor that causes induction of a-sma expression.
  • the Examples set out details of in vitro studies (in human dermal fibroblasts), which illustrate the ability of compositions of the invention to block this increase in a-sma expression. This indicates that the decorin incorporated in the ocular hydrogel compositions of the invention is able to effectively block the activity of fibrotic growth factors (such as TGF-b) despite the absence of collagen and/or fibrin in the exemplary compositions used.
  • fibrotic growth factors such as TGF-b
  • Fibrosis is also associated with the expression and deposition of ECM constituents at a site of injury.
  • the amount of ECM deposited may be increased in scarring, and the arrangement of the ECM may be different from that found in undamaged comparator tissue.
  • the data presented in the Examples illustrate that treatment using compositions of the invention gives rise to tissues in which the arrangement of ECM components more closely resembles that of unwounded tissue, thus illustrating the utility of these compositions in the inhibition of scarring.
  • compositions of the invention comprising anti-fibrotic decorin, may be able to achieve an inhibition of fibrosis in the eye by at least 5% as compared to a suitable control agent.
  • a suitable anti-fibrotic agent may be able to achieve an inhibition of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, as compared to a suitable control agent.
  • An anti-fibrotic agent suitable for incorporation in a composition of the invention may be able to achieve substantially total inhibition of scarring as compared to a suitable control agent.
  • compositions of the invention or methods of treatment using such compositions, to inhibit scarring in the eye may achieve an inhibition of at least 5% as compared to a suitable control.
  • such medical uses or methods of treatment may achieve an inhibition of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, as compared to a suitable control.
  • the medical uses or methods of treatment of the invention may achieve substantially total inhibition of scarring as compared to a suitable control.
  • a suitable control for assessment of the ability of a composition of the invention to inhibit scarring in the eye may be provided by the recognised standard of care, or an experimental proxy thereof.
  • compositions of the invention intended for medical use, or use in methods of treatment may comprise a further active agent, in addition to the anti-fibrotic agent decorin.
  • a suitable further active agent may be selected with reference to the intended medical use.
  • a suitable further active agent may be selected from the group consisting of: a further anti-fibrotic agent; an anti-infective agent; and an anti-inflammatory agent.
  • a composition of the invention may suitable comprise more than one active agent.
  • this may be more than one active agent within a particular class of active agents (e.g. two or more anti-fibrotic agents), or a combination of agents selected from two or more different classes (e.g. anti-fibrotic decorin and an anti-infective agent, or an anti- fibrotic decorin and an anti-inflammatory agent).
  • an anti-infective agent suitable for incorporation as an active agent in a composition of the invention may be an anti-microbial agent.
  • an anti-viral agent such as an anti-viral agent, an anti-fungal agent, or anti-helminth agent.
  • a suitable anti-infective agent may be an antibiotic, such as gentamicin.
  • antibiotics such as gentamicin.
  • a composition of the invention comprising an anti-infective agent may be used in methods for the prevention and/or treatment of infection. Accordingly, it will be appreciated that such a composition may be administered to a subject in need of prevention and/or treatment of infection. A subject in need of such prevention and/or treatment may be one that has microbial keratitis.
  • An anti-inflammatory agent for incorporation as an active agent in a composition of the invention may be selected from the group consisting of: a steroid, such as a corticosteroid (for example prednisolone); a non-steroidal anti-inflammatory drug (NSAID), such COX-1 and/or COX-2 enzyme inhibitor; an anti-histamine, such as an H1 receptor antagonist; interleukin-10; pirfenidone; an immunomodulatory agent; and a heparin-like agent.
  • a steroid such as a corticosteroid (for example prednisolone); a non-steroidal anti-inflammatory drug (NSAID), such COX-1 and/or COX-2 enzyme inhibitor
  • an anti-histamine such as an H1 receptor antagonist
  • interleukin-10 interleukin-10
  • pirfenidone an immunomodulatory agent
  • heparin-like agent heparin-like agent
  • a composition of the invention comprising an anti-inflammatory agent may be used in methods for the prevention and/or treatment of inflammation. Accordingly, such a composition may be administered to a subject in need of prevention and/or treatment of inflammation. Suitably, the subject may be one having or at risk of developing chronic inflammation or acute inflammation. Merely by way of example, inflammation may be caused by microbial keratitis.
  • composition of the invention may comprise decorin for use in combination with the anti- infective agent gentamicin, and the anti-inflammatory agent prednisolone.
  • a composition of this sort may comprise decorin, prednisolone and gentamicin.
  • Such compositions of the invention are suitable for use in the inhibition of scarring associated with microbial keratitis, as illustrated by the data set out in the Examples.
  • a composition of the invention incorporates the anti-fibrotic agent decorin in a therapeutically effective amount.
  • Therapeutically effective amounts of anti-fibrotic agents, such as decorin, are discussed in further detail below.
  • a composition of the invention may also comprise a further active agent in a therapeutically effective amount.
  • a therapeutically effective amount of decorin or a further active agent will be able to achieve a desired clinical outcome either in a single administration, or as part of a course of treatment comprising multiple incidences of administration.
  • the skilled person will be well aware of suitable protocols and procedures for the calculation of therapeutically effective amounts of active agents of various sorts.
  • an active agent may be incorporated in a composition of the invention at a concentration of between 0.1 ng/ml_ and 10mg/ml_.
  • an active agent may be incorporated in a composition of the invention at a concentration of between 1 ng/ml_ and 5mg/ml_, between 10ng/ml_ and 2.5mg/ml_, or between 20ng/ml_ and 1mg/ml_, between about 0.1 pg/mL and 0.5pg/ml_, suitably about 0.24pg/ml_.
  • Anti-fibrotic agents are agents that are able to bring about an inhibition of scarring in a body site to which they are provided.
  • the inhibition of scarring is considered more generally elsewhere in the specification.
  • Decorin incorporated in the ocular hydrogel compositions of the invention, is an example of an anti-fibrotic agent, and many other anti-fibrotic agents are known to those skilled in the art. Accordingly, the skilled person will be readily able to identify anti-fibrotic agents that may beneficially be incorporated in compositions of the invention for use in the inhibition of scarring. The following provides a non-exclusive list of examples of anti-fibrotic agents suitable for such uses.
  • Suitable anti-fibrotic agents may be selected from the group consisting of: anti- fibrotic extracellular matrix (ECM) components; anti-fibrotic growth factors (which for purposes of the present disclosure should be taken as also encompassing anti-fibrotic cytokines, chemokines, and the like); and inhibitors of fibrotic agents, such as function blocking antibodies.
  • ECM extracellular matrix
  • anti-fibrotic growth factors which for purposes of the present disclosure should be taken as also encompassing anti-fibrotic cytokines, chemokines, and the like
  • inhibitors of fibrotic agents such as function blocking antibodies.
  • Antibodies are useful in disrupting certain cellular activities by binding to cell signalling agents and thereby blocking functions caused by the agents’ activity. Examples of such activities that may be blocked include: cell proliferation, cell migration, protease production, apoptosis and anoikis.
  • suitable blocking antibodies may be able to bind one or more of the following groups of cell signalling agents: ECM components, growth factors, cytokines, chemokines or matrikines.
  • Decorin is an example of an anti-fibrotic ECM component.
  • the decorin may be human decorin.
  • the decorin may be human recombinant decorin.
  • An example of a human recombinant decorin that may be incorporated in the compositions of the invention is that produced and sold by Catalent Pharma Solutions, Inc., under the name“GalacorinTM”.
  • Decorin for incorporation in a composition of the invention may be a full-length naturally occurring version of this proteoglycan.
  • compositions of the invention may employ anti-fibrotic fragments or anti-fibrotic variants of naturally occurring decorin.
  • Naturally occurring decorin is a proteoglycan.
  • the proteoglycan (comprising both the core protein and glycosaminoglycan chains), or its fragments, may be used in the hydrogel compositions of the invention.
  • the inventors have demonstrated that the core protein alone (without glycosaminoglycan chains) is sufficient to inhibit scarring in the eye.
  • references to decorin (or fragments or variants thereof), in the present specification may alternatively be construed as directed to the core protein without glycosaminoglycan chains.
  • a suitable anti-fibrotic fragment of decorin may comprise up to 50% of the full- length, naturally occurring molecule, up to 75% of the full-length, naturally occurring molecule, or up to 90% of the full-length, naturally occurring molecule.
  • a suitable anti-fibrotic fragment of decorin may comprise the TGF ⁇ -binding portion of decorin.
  • an anti-fibrotic variant of decorin will differ from the naturally occurring proteoglycan by the presence of one or more mutations in the amino acid sequence of the core protein. These mutations may give rise to additions, deletions, or substitutions of one or more amino acid residues present in the core protein.
  • a suitable anti-fibrotic variant of decorin suitable for incorporation in the compositions of the invention may comprise at least 1 , at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, or at least 20 mutations as compared to the amino acid sequence of the naturally occurring core protein.
  • references herein to decorin in connection with the incorporation of this agent in the compositions of the invention, should also be taken as encompassing the use of anti-fibrotic fragments or anti-fibrotic variants of decorin.
  • decorin constitutes the only ECM component present in a composition of the invention.
  • the decorin may be the only anti-fibrotic active agent incorporated in an ocular hydrogel composition in accordance with the invention.
  • Anti-fibrotic growth factors suitable for incorporation in compositions of the invention include those selected from the group consisting of: transforming growth factory, platelet derived growth factor AA, insulin-like growth factor-1 , epidermal growth factor, fibroblast growth factors (FGF) 2, FGF7, FGF10, FGF22, vascular endothelial growth factor A, keratinocyte growth factor, and hepatocyte growth factor.
  • FGF fibroblast growth factors
  • Inhibitors of fibrotic agents represent suitable examples of further anti-fibrotic agents that may be incorporated in the compositions of the invention.
  • Examples of such inhibitors include agents that bind to, and thereby block, the activity of a fibrotic agent.
  • Examples of such inhibitors include function blocking antibodies or soluble fragments of cell receptors by which the fibrotic agent induces cell signalling.
  • Other examples of such inhibitors include agents that prevent expression of the fibrotic agent. Examples of these sorts of inhibitors include those selected from a group consisting of: anti-sense oligonucleotides, and interfering RNA sequences.
  • a composition of the invention suitable for use in the inhibition of scarring will incorporate an anti-fibrotic agent in a therapeutically effective amount.
  • a therapeutically effective amount will be able to inhibit scarring either in a single administration, or as part of a course of treatment comprising multiple incidences of administration. Details of how inhibition of scarring may be assessed, and so how a therapeutically effective amount may be calculated or recognised, are considered above.
  • the composition of the invention may comprise decorin at a concentration of between 0.1 ng/ml_ and 10mg/ml_, between 1 ng/ml_ and 5mg/ml_, between 10ng/ml_ and 2.5mg/ml_, between 20ng/ml_ and 1 mg/ml_, between about 0.1 pg/mL and 0.5pg/ml_, suitably about 0.24pg/ml_.
  • compositions of the invention are suitable for topical administration to the eye.
  • topical administration is taken to relate to direct administration of the composition to a surface of the eye.
  • a composition of the invention suitable for such topical administration may be referred to as a topical ocular composition of the invention.
  • Topical compositions of the invention may be for administration to sites of infection or injury on the surface of the eye including, but not limited to: infections, abrasions, incisions, excisions, burns, and puncture wounds.
  • topical compositions of the invention may be for administration to the cornea.
  • topical compositions may be formulated in manners conventional for use in such contexts.
  • a suitable topical composition may be formulated such that it does not induce irritation or inflammation of an infected or injured area to which it is administered.
  • the inventors have provided a novel eye drop system for the sustained delivery of a potent anti-scarring molecule (hrDecorin).
  • the novelty of this eye drop lies in the method of structuring during manufacture, which creates a material that can transition between solid and liquid states, allowing retention in a dynamic environment being slowly removed through blinking.
  • applying the eye drop resulted in reductions of corneal opacity within 16 days.
  • the addition of hrDecorin resulted in scarless restoration and corneal integrity, as shown by complete re- epithelialization and reductions in aSMA, fibronectin and laminin.
  • This drug delivery system is an ideal non-invasive anti-fibrotic treatment for patients with microbial keratitis, potentially without recourse to surgery saving the sight of many in the developing world, where corneal transplantation may not be available.
  • the present inventors have provided report a new class of eye drop material that allows for prolonged retention of a therapeutic on the surface of the eye, while being gradually cleared through the blinking process.
  • the material is formed through the shearing of a gellan-based hydrogel, a material that is currently used in dilute form to thicken eye drops (e.g. Timoptol) during the gelation process.
  • the application of shear prevents the formation of a continuous polymeric network and results in the formation of interacting particles that can exhibit spherical and ribbon-like morphology. Following shear-processing, these particles interact and form a continuous structure when the solution is at rest.
  • a fluid-gel eye drop has been developed which can be loaded with decorin, to provide localized drug delivery and retention at the surface of the eye.
  • the material combines structured gellan gum with the proteoglycan, decorin.
  • the FDA approved polymer FDA reference number 172.665
  • hrDecorin provides a rapid route to translation into the clinic.
  • this study investigated the effects of fluid gel, with and without hrDecorin, on corneal opacity, wound healing and fibrosis within a well-established murine model of Pseudomonas keratitis, as a precursor to clinical application for the management of severe bacterial infection.
  • Processing of the fluid gel involves passing a polymer solution, gellan, through a jacketed pin-stirrer, where it experiences high levels of shear whilst being forced (thermally) through its sol-gel transition (Fig 1a). This restricts the long-range ordering normally observed in the formation of quiescent gels, restricting growth of the gel nuclei to discrete
  • microstructures within the eye drop prepared in this way have been
  • the gellan-based eye drop system was formulated for drug delivery with the candidate anti-fibrotic agent, hrDecorin, used for our studies.
  • the rate of release of hrDecorin from the eye drop system was almost linear over time (Fig 2a).
  • Turbidity was used as a measurement of fibrillogenesis (formation of large, disorientated collagen fibers), shown as a function of the hrDecorin (Figs 2b&c). It was evident that hrDecorin played a key role in the kinetics of fibril formation, slowing the onset of fibrillogenesis, and also reaching an equilibrium much faster (Fig 2b).
  • mice treated with the standard of care with hrDecorin fluid gel showed significantly (p ⁇ 0.001) lower opaque areas (1 9 ⁇ 0.3 mm 2 ) compared with eyes treated with Gentamicin and Prednisolone only (3.5 ⁇ 0.4 mm 2 ).
  • Epithelial stratification/maturation together with stromal thickness were chosen as outcome measures to assess corneal re-epithelialization, and to observe thickening of the stroma from edema and cellular infiltrates (as markers of infection).
  • Pseudomonas infection severely disrupted the corneal structure, with an averaged increased corneal thickness of 218.7 ⁇ 24 mm after the infection on day 2 compared to naive corneal thickness values of 129.3 ⁇ 10.7 mm.
  • the infected corneas at day 2 had thinner epithelial layers compared to normal intact controls (19.2 ⁇ 2.1 mm vs 35.5 ⁇ 1.7 mm; Figs 4a & b).
  • IR Immunoreactivity
  • stromal IR aSMA stromal IR aSMA remained elevated at day 16, at 32.7 ⁇ 6.1% in eyes treated with standard of care only.
  • the level of stromal aSMA IR was significantly lower at day 16, 13.4 ⁇ 2.9% and 2.0 ⁇ 0.4% respectively, suggesting less myofibroblast activation within the corneal stroma.
  • the hrDecorin fluid gel was most effective at keeping the aSMA IR levels low, resulting in similar values to the intact cornea, suggesting that the addition of hrDecorin in the fluid gel had an added beneficial effect on myofibroblast differentiation vs the fluid gel alone (Fig 5a).
  • IR laminin levels of IR laminin (Fig 5c) demonstrated that the infection increased levels of laminin when compared with the intact cornea, from 2.15 ⁇ 0.6% in the intact to 16.3 ⁇ 4.6% in the infection group at day 2. Levels of IR laminin continued to rise by day 16 after Gentamicin and Prednisolone treatment to 42.5 ⁇ 8.2%. Similar to the Gentamicin and Prednisolone group, average levels of IR laminin remained high on day 16 after treatment with the fluid gel, with IR laminin levels at 38.0 ⁇ 12.0%.
  • the structured or“fluid-gel” formed from gellan provides a pivotal advance since it enables the sustained delivery of molecules such as hrDecorin capable of preventing scarring and obviating the need for invasive surgical repair strategies.
  • the major advantage of the gellan fluid-gel is its capacity to transition between solid and liquid states as it passes through the applicator and solidifies on the surface of the cornea. This unique set of properties originates from the microstructure of the material, which consisted of ribbons and particles that weakly interact with one another at zero shear. These interactions are broken by the application of shear and reform following its removal. In this way, the material may then be gradually cleared from the ocular surface through the natural blinking mechanism.
  • the mouse model of P. aeruginosa keratitis provides a robust, clinically relevant means of evaluating the anti-scarring capacity of the hrDecorin loaded fluid gel against the current standard of care for pseudomonas infection (Gentamicin and Prednisolone) [43] .
  • P. aeruginosa invades the corneal epithelial cells disrupting the natural healing responses with transformation of corneal fibroblast into corneal myofibroblasts leading to a fibrogenic Topical administration of the eye drops either with or without the hrDecorin resulted in reduced levels of corneal opacity after 7 and 10 days of eye drop treatments, with the addition of hrDecorin displaying an evident further advantage.
  • the effects of the fluid gel only treatment were not expected as the initial in vitro studies demonstrated that this carrier appeared inert.
  • the therapeutic efficacy of fluid gel alone may be due to the formation of a permissive microenvironment in the damaged cornea, where the occlusive effect of the gel ribbons (that entwine to form a barrier around the wound) provided a therapeutic bandage that prevent biomechanical trauma caused by blinking over the ulcerated eye. It may also have sequestered Prednisolone and Gentamicin within its structure, enhancing retention of the therapeutic substances to the ocular surface, thereby improving bioavailability similar to the prosthetic replacement of the ecosystem (PROSE (TM) device) but with the added advantage of being resorbable.
  • hrDecorin treated eyes exhibited the most improved restoration to normal anatomy, with a reduction in stromal edema, thickness and extracellular matrix deposition, coupled with improved epithelial morphology.
  • the reduction in fibrotic markers by hrDecorin has been previously demonstrated across numerous animal models; modulating a range of growth factors (e.g.
  • VEGF vascular endothelial growth factor
  • IGF-1 IGF-1
  • EGF EGF
  • PDGF vascular endothelial growth factor
  • T ⁇ Rb signaling via SMAD 2 and 3 pathways preventing differentiation of corneal fibroblasts.
  • MMPs matrix metalloproteinase
  • T ⁇ Rb signaling via SMAD 2 and 3 pathways preventing differentiation of corneal fibroblasts.
  • MMPs matrix metalloproteinase
  • TRIP tissue inhibitors of metalloproteinase
  • the effects of the fluid gel alone on the damaged corneal surface suggests an influence over the endogenous growth factors, an effect that is enhanced by the addition of hrDecorin.
  • the fluid-gel may aid corneal healing through several mechanisms: firstly, the unique viscoelastic properties of the fluid gel acts as a liquid that self-structures upon the ocular surface to form a semi-solid occlusive therapeutic dressing for unperturbed healing to take place; secondly, helical domains formed during the gelation of the fluid gel may provide a mimetic scaffold for endogenous decorin to bind, sequestering key growth factors e.g.
  • the fluid gel matrix comprising primarily of water (99.1%), creates a gradient driven diffusion of cytokines away from the wound site, again resulting in a restoration of the natural equilibria needed to prevent fibrosis.
  • the inventors have seen two different responses in the presence of the fluid gel and the Decorin fluid gel, and in future studies it will be important to tease apart the mechanism for each.
  • the fluid gel alone is providing a protective barrier whilst potentially influencing inflammatory cell and fibroblast behavior in a manner that we do not yet understand.
  • the fluid gels are facilitating regeneration of the corneal epithelium leading to wound closure.
  • We are hypothesizing that the fluid gel is affecting the limbal epithelium stem cell niche by promoting proliferation and differentiation, which may be dysregulated in the disease situation as well as providing a therapeutic bandage to aide stromal repair.
  • a novel eye drop technology can be used to provide sustained delivery of anti-fibrotic drugs like hrDecorin topically to the cornea in a clinically relevant murine model of fibrosis associated with bacterial keratitis.
  • the eye drop enabled the hrDecorin to remain in contact with the surface of the eye for long enough and at sufficient titers to significantly reduce corneal scarring.
  • this study has demonstrated that the unloaded fluid gel also possesses healing effects in its own right, suggested to arise through its intrinsic material microstructure and subsequent properties.
  • the aim of this study was to explore the use of a novel fluid gel to deliver decorin to the ocular surface in order to reduce corneal opacity and scarring post-bacterial keratitis.
  • the study was split into three evaluation stages: (i) material properties relating to ease of eye drop application, (ii) in vitro assessment of bioactivity of the formulated hrDecorin and (iii) anti-scarring efficacy of the fluid gel with/without hrDecorin in vivo, using a mouse model of Pseudomonas keratitis (once the eyes were sterilized after infection) in comparison to the current standard of care.
  • Fluid gels were produced by first dissolving low acyl gellan gum (Kelco gel CG LA, Azelis, UK) in deionized water. Gellan powder was added to deionized water at ambient temperature in the correct ratio to result in a 1% (w/v) solution. The sol was heated to 70 °C under agitation, on a hotplate equipped with a magnetic stirrer, until all the polymer had dissolved. Once dissolved, gellan sol was added to the cup of a rotational rheometer (AR- G2, TA Instruments, UK) equipped with cup and vane geometry (cup: 35 mm diameter, vane: 28 mm diameter). The system was then cooled to 40 °C.
  • a rotational rheometer AR- G2, TA Instruments, UK
  • hrDecorin (GalacorinTM; Catalent, USA) in PBS (4.76 mg/ml) and aqueous sodium chloride (0.2 M) was then added to result in final concentrations of 0.9% (w/v) gellan, 0.24 mg/ml hrDecorin and 10 mM NaCI. Following this, the mixture was cooled at a rate of 1 °C/min under shear (450 /s) to a final temperature of 20 °C. The sample was then removed and stored at 4 °C until further use. In the case of fluid gels without hrDecorin, ratios were adjusted so that the final eye drop had a composition of 0.9% (w/v) gellan, 10 mM NaCI.
  • Microscopy For transmission microscopy samples were first diluted using polyethylene glycol 400 (PEG400) at a ratio of 1 :4 (eye drop to PEG400). Following this, samples were analyzed using an Olympus FV3000. Images were processed using ImageJ (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA).
  • Viscosity profiles were obtained using an AR-G2 (TA Instruments, UK) rheometer equipped with sandblasted parallel plates (40 mm, 1 mm gap height) at 20 °C. An equilibrium of 2 minutes was used to ensure constant test temperature. Following this, time dependent ramps up and down were applied ranging from 0.1 to 600 /s (3 minutes sweep times). Recovery profiles were obtained using the same apparatus, under single frequency. The sample underwent rejuvenation by shearing at 600 /s for 10 s. Following this, storage and loss (G’, G” respectively) were monitored at 1 Hz, 0.5% strain. The cross over point was used as the point at which the sample started to act like a viscoelastic solid. hrDecorin release from the fluid gel
  • hrDecorin release was determined cumulatively, by placing 1 ml of the fluid gel containing hrDecorin in a 6 well plate. Then 2 ml of DMEM was placed over the sample and the plates were incubated at 37 °C. At each time point, the media was removed for measurement of hrDecorin, and replaced with fresh media. Decorin release was quantified using an ELISA specific for human Decorin (R&D systems, Minneapolis, USA) in accordance with the manufacturer’s protocol.
  • Collagen fibrillogenesis For the dose response curves, 75 pi of PBS was added to each well of a 96 well plate kept on ice. Varying hrDecorin doses were prepared by adding 400mg/ml of hrDecorin to the first well and subsequently serial diluting (2-fold dilution) across the plate. Following dilution, a further 150 mI of PBS buffer was added to each well. Then, 75 mI of collagen type I (rat tail; Corning, UK) (800 mg/ml) was added to each well and incubated for 2 hours at 37 °C. Subsequent absorbance readings were taken using a 405 nm plate reader.
  • Each assay consisted of duplicate blank controls, and triplicate standard dilutions followed by triplicate sample dilutions.
  • Kinetics of fibril formation were determined using a similar setup as the dose response, without serial dilution; incubating the samples within the plate reader, and taking data points every 2 minutes.
  • the treatment administration regimes for the in vivo Pseudomonas model are shown in Figure 6.
  • Groups of naive intact and infected corneas taken at day 2 were also included in the experimental plan.
  • P. aeruginosa PA01 strain was cultured in high salt LB (10 g of tryptone, 5 g of yeast extract, and 11.7 g of NaCI per L, supplemented with 10 mM MgCL and 0.5 mM CaCL) at 37 °C for 18 hours. Sub-cultures were derived at an optic density (OD) of 0.2 (OD 650nm approx. 1x10 8 CFU/ml). P. aeruginosa were washed (x3) in PBS, centrifuged at 300 rmm for 5 minutes and re-suspended in PBS at a density of 1x10 5 CFU/2.5pl.
  • OD optic density
  • mice C57BL/6 mice (Jackson Laboratory, CA, USA) were housed in pathogen-free conditions, given free access to food and water and were maintained according to the ARRIVE guidelines, the ARVO statement for the use of animals in ophthalmic and vision research and also adhered to guidelines set out by the University of California, Irvine.
  • mice were anaesthetized and one corneal epithelium was abraded with 3x1 mm parallel scratches using a 26 G needle and inoculated with 2.5 pi P. aeruginosa (1x10 5 CFU) (strain PA01) 64 ⁇ 65 . Mice remained sedated for 2 hours post-inoculation to permit penetration of the infection into the eye, and placed in recovery.
  • mice were treated with 5 mI of Gentamicin (1.5%, QEHB Pharmacy, Birmingham, UK) every 2 hours for a 12-hour period, to sterilize the infection. After a further 12 hours, mice were administered eye drops (5 mI of each compound) every 4 hours between 8am and 8mm for a further 13 days depending on their treatment group: (1) Gentamicin + Prednisolone (0.5%, QEHB Pharmacy), (2) Gentamicin + Prednisolone + Fluid gel, or (3) Gentamicin + Prednisolone + Fluid gel with hrDecorin. Mice were examined for corneal opacification, ulceration and perforation.
  • Enucleated eyes for IHC were post-fixed by immersion in 4% PFA in PBS overnight at 4 °C before cryoprotection using increasing concentrations of sucrose in PBS (10%, 20%, and 30%; Sigma) for 24 hours each at 4 °C. Eyes were then embedded in optimal cutting temperature (OCT) embedding medium (Thermo Shandon, Runcorn, UK) in peel-away mold containers (Agar Scientific, Essex, UK) and later sectioned in the parasagittal plane at -22 °C using a cryostat microtome (Bright, Huntingdon, UK) at a thickness of 15 mm, and placed onto Superfrost slides (Fisher Scientific, USA).
  • OCT optimal cutting temperature
  • Non-specific antibody binding sites in tissue sections were blocked for 30 minutes using 0.5% BSA, 0.3% Tween-20 (all from Sigma), and 15% normal goat serum (Vector Laboratories, Peterborough, UK) in PBS before incubating overnight in 4 °C in primary antibody (aSMA, Laminin and fibronectin; 1 :200; all from Sigma) again followed by washing 3x5 minutes, and incubating for 1 hour at room temperature with a secondary antibody (Goat anti-mouse Alexa Fluor 488 1 :500, Goat anti-mouse Alexa Fluor 594 1 :500, Molecular Probes, Paisley, UK). Sections were then washed for 3x5 minutes and mounted in Vectorshield mounting medium containing DAPI (Vector Laboratories). Control tissue sections incubated with secondary antibody alone were all negatively stained.
  • aSMA Laminin and fibronectin; 1 :200; all from Sigma
  • Table 2 Table of biopolymers, both polysaccharide and protein based, with the potential to be processed into microgel suspensions using a shearing technique. Addition information of charge, isoelectric point (pi) for proteins, gelling mechanism and optical clarity has been given - if transparent, the gels has potential application in ophthalmic devices, however this is not limited to.
  • Optimal eye drop viscosity was sought via two main methods: rheological characterisation of current, commercial eye drops/ointments, and consultation with ophthalmic clinicians. Characterisation of the commercial eye products highlighted a large range of viscosities across both eye drops and eye ointments used to medicate conditions such as dry eye; where optimally long retention times are required. Viscosities were collected and compared at 1 s-1 (chosen as a value within initial stages of shear thinning, so as to avoid artefacts for the apparatus) (Table 2 and Fig 6 (section A.1.)), highlighting similar viscosities between products as a function of the polymer they were predominately made from: paraffin, carbomer and biopolymer based.
  • Table 1 Table of viscosities derived at 1 s 1 for commercially available eye drops/ointments.
  • the system should exhibit shear thinning behaviour.
  • Table 2 Summary of potential viscosities as found at 1 s -1 (20 °C) for eye drop formulations.
  • Table 4 Summary of potential elastic moduli as found within the linear viscoelastic region (LVR) at 1 Hz (20 °C) using a strain sweep for eye drop formulations.
  • the material properties of the formulations are governed by the concentration of initial polymer in the product. Therefore, the upper and lower material properties were used to evaluate the material formulations, providing upper and lower limits for polymer concentrations. As all systems exhibited shear thinning behaviours limits were solely based on fulfilling the criteria for both viscosity (at 1 s _1 ) and elastic behaviour at rest. Thus, the maximum range of:
  • 0.1 to 5.0 wt.% e.g. 0.1 to 3.5 wt.% or 0.5 to 2.5%) (w/v) has been set for eye drop formulations, with values within those found for commercially available products. This have been narrowed to fall within the clinician’s advice to: 0.5 to 1.5% (w/v) with the optimised formulation consisting of:
  • PBS The addition of PBS can be used to manipulate the pH of the system. In these cases,
  • the thermal processing within the manufacture are key to formation of a gel.
  • the thermal parameters are divided into two sections: the processing temperatures, and the rate of cooling.
  • the inlet and outlet are key to make sure the polymer is in a sol prior to processing and exits at a temperature below the gelling transition.
  • the inlet temperature was set as close to the gelling temperature as possible, due to the protein active denaturing at higher temperatures. Therefore, this was set to 40 °C.
  • the key aspect to the inlet temperature is to keep it above the gelling temperature, to prevent early gelation and blockages.
  • the function of the outlet temperature is to ensure ordering/structuring of the polymer has completed prior to storage. This prevents aggregation during stage and heterogeneous suspensions forming. As such, for gellan this temperature has been defined as 20 °C, allowing the polymer to pass though the gelation process.
  • the exit temperature is therefore controlled by the jacket of the mill, which is set to provide sufficient cooling during the process. This can be changed resulting in various cooling rates.
  • the cooling rate during the sol-gel transition is known to be very important in regard to the final material properties; as higher cooling rates result in rapid formation of structures and weaker overall moduli. This was observed for the microgel suspensions, however, only at higher polymer concentrations. It was observed that for the optimal eye drop formulation, no changes in material properties were observed suggesting that a large range of parameters could be used:
  • Table 10 Summary of processing rates for the gellan eye drop formulation.
  • Table 11 Summary of potential pH’s for eye drop formulations.
  • Table 12 Summary of potential pH’s for eye drop formulations.
  • NaCI (0.2 M) was prepared through the addition of dry crystals (1.16 g) to deionised water (100 ml) using a volumetric flask. The NaCI was then allowed to dissolve using an inverting technique to aid the process. Once fully dissolved the solution was kept at ambient conditions until further use.
  • Gellan sols were prepared by dissolving powdered polymer into water/NaCI solution at varying ratios so that the final concentrations post-processing were equal to 0.5, 0.9, 1.35, 1.8 and 2.35% (w/v).
  • gellan powder was weighed out (2.5, 4.5, 6.75, 9.0 and 11.75 g) and added to 450 ml of deionised water. The mixture was allowed to heat to 95 °C under agitation, allowing the polymer to dissolve. Once fully dissolved, 25 ml of NaCI stock solution (0.2 M) was added to the solution resulting in a 10 mM concentration post-processing. The sol was then allowed to reach thermal equilibrium at 95 °C before processing.
  • MS were prepared using a jacketed pin mill set to 20 °C.
  • Gellan sols were pumped using a peristaltic pump into the pin mill at 3 ml/min so that it entered the processing chamber at 40 °C.
  • water was pumped into the gellan stream (at a rate of 0.16 ml/min) so that they impinged, diluting the gellan sol to the final concentrations (0.5, 0.9, 1.35, 1.8 and 2.35% (w/v), 10 mM NaCI).
  • the mixture was then cooled under shear (500 rmm or 1000 rmm) as it passed through the milling unit. On exiting, at 20 °C, the gel was packaged and stored at 4 °C until further testing.
  • a rheometer (TA, AR-G2) equipped with a sandblasted parallel plate (40 mm diameter, 1 mm gap height) was used to test all samples, at 20 °C. esults are shown in Figures 7 to 9
  • Amplitude sweeps were obtained in strain controlled mode over a range of 0.1 to 100.0 %. Samples were loaded into the instrument and upper geometry lowered. Once trimmed, the sample was left to equilibrate at 20 °C prior to testing. Measurements were obtained at 1 Hz in a logarithmic fashion.
  • Viscosity profiles for the samples were obtained using a continuous ramp. Samples were loaded into the instrument and upper geometry lowered. Once trimmed, the sample was left to equilibrate at 20 °C prior to testing. Increasing shear was applied to the sample in rate controlled mode, between 0.1 and 600 s _1 over a 3-minute ramp, with data points obtained in a logarithmic fashion.
  • the rheology of the suspensions also closely correlates to those of emulsions; where increasing the phase volume of the droplets or particles (in this case) results in closer proximity and an increase in both the elastic nature (G’) and viscosity of the systems. In this case, increasing the polymer concertation results in a larger number of particles, until a maximum packing fraction is reached. Above this, no further changes in material properties are seen.
  • LVR linear viscoelastic region
  • shear thinning behaviour is also vital for both application and elimination, allowing the suspensions to easily flow upon liquefaction. Shear thinning was observed across all systems, irrespective of polymer concentration (Fig. 4). A high degree of shear thinning, arising through the breakdown of inter-particle interactions and alignment in flow allows the systems to be easily applied through a nozzle (syringe, single use applicator etc.); where small pressures result in high levels of shear.
  • NaCI (0.1 , 0.2, 0.4 and 0.8 M) were prepared through the addition of dry crystals (0.58, 1.16, 2.32 and 4.64 g) to deionised water (100 ml) using a volumetric flask. The NaCI was then allowed to dissolve using an inverting technique to aid the process. Once fully dissolved the solutions were kept at ambient conditions until further use.
  • Gellan solutions were prepared by dissolving powdered polymer into water/NaCI solution so that the final concentrations post-processing were equal to 0.9% and 1.8% (w/v).
  • gellan powder was weighed out (4.5 g, 9.0 g) and added to 450 ml of deionised water. The mixture was allowed to heat to 95 °C under agitation, allowing the polymer to dissolve. Once fully dissolved, 25 ml of NaCI stock solution (either 0.1 , 0.2, 0.4 and 0.8 M) was added to the solution resulting in a 5, 10, 20 or 40 mM concentration post-processing. The sol was then allowed to reach thermal equilibrium at 95 °C before processing.
  • MS were prepared using a jacketed pin mill set to 20 °C.
  • Gellan sols were pumped using a peristaltic pump into the pin mill at 3 ml/min so that it entered the processing chamber at 40 °C.
  • water was pumped into the gellan stream (at a rate of 0.16 ml/min) so that they impinged, diluting the gellan sol to the final concentrations (0.9% and 1.8% (w/v); 5, 10, 20 or 40 mM NaCI).
  • the mixture was then cooled under shear (1000 rmm) as it passed through the milling unit. On exiting, at 20 °C, the gel was packaged and stored at 4 °C until further testing.
  • a rheometer (TA, AR-G2) equipped with a sandblasted parallel plate (40 mm diameter, 1 mm gap height) was used to test all samples, at 20 °C.
  • Amplitude sweeps were obtained in strain controlled mode over a range of 0.1 to 100.0 %. Samples were loaded into the instrument and upper geometry lowered. Once trimmed, the sample was left to equilibrate at 20 °C prior to testing. Measurements were obtained at 1 Hz in a logarithmic fashion.
  • Viscosity profiles for the samples were obtained using a continuous ramp. Samples were loaded into the instrument and upper geometry lowered. Once trimmed, the sample was left to equilibrate at 20 °C prior to testing. Increasing shear was applied to the sample in rate controlled mode, between 0.1 and 600 s _1 over a 3-minute ramp, with data points obtained in a logarithmic fashion.
  • salts play a vital role in the gelation of many polymers, including gellan.
  • Salt type particularly valency (mono, di, tri etc.) are key to the resultant gel properties; typically, increasing the valency increases the gel strength, as more bridges are formed between polymers.
  • di-valent ions e.g. Ca 2+
  • mono-valent ions such as Na + can be used to strengthen the junction sites between helices, forming the 3-dimentional gel structure. Therefore, resultant gel strength is a function of concentration of salt, also termed cross-linker, added.
  • NaCI (0.2 M) was prepared through the addition of dry crystals (1.16 g) to deionised water (100 ml) using a volumetric flask. The NaCI was then allowed to dissolve using an inverting technique to aid the process. Once fully dissolved the solutions were kept at ambient conditions until further use.
  • Gellan solutions were prepared by dissolving powdered polymer into water/NaCI solution so that the final concentrations post-processing were equal to 0.9% and 1.8% (w/v).
  • gellan powder was weighed out (4.5 g, 9.0 g) and added to 475 ml of deionised water. The mixture was allowed to heat to 95 °C under agitation, allowing the polymer to dissolve. Once fully dissolved, 25 ml of NaCI stock solution (0.2 M) was added to the gellan sol, resulting in a 10 mM final concentration. The sol was then allowed to reach thermal equilibrium at 95 °C before processing.
  • MS were prepared using a jacketed pin mill, whereby the jacket temperature and the residence time within the mill were altered to result in cooling rates of 1 , 3 and 6“Cmin -1 .
  • the jacket was set to 5 °C and with a flow rate of 20 mimin 1 , the temperature of the fluid at the inlet was 46 and outlet 16, the residence time at this rate was 5 minutes, thus the cooling rate was equal to 6“Cmin -1 .
  • the gel was packaged and stored at 4 °C until further testing.
  • a rheometer (TA, AR-G2) equipped with a sandblasted parallel plate (40 mm diameter, 1 mm gap height) was used to test all samples, at 20 °C.
  • Amplitude sweeps were obtained in strain controlled mode over a range of 0.1 to 100.0 %. Samples were loaded into the instrument and upper geometry lowered. Once trimmed, the sample was left to equilibrate at 20 °C prior to testing. Measurements were obtained at 1 Hz in a logarithmic fashion.
  • Viscosity profiles for the samples were obtained using a continuous ramp. Samples were loaded into the instrument and upper geometry lowered. Once trimmed, the sample was left to equilibrate at 20 °C prior to testing. Increasing shear was applied to the sample in rate controlled mode, between 0.1 and 600 s _1 over a 3-minute ramp, with data points obtained in a logarithmic fashion.
  • Cooling plays a key role in the formation of gellan hydrogels, forcing the polymers through a random coil to helix transition.
  • the effects of cooling rate on the formation of fluid gels was studied to evaluate related changes in material response. It was observed that at the lower polymer concentration (0.9% (w/v)) the cooling rate had little effect on both the degree of elasticity within the system and overall viscosity. However, at higher concentrations (1.8% (w/v)), the cooling rate has a much more pronounced effect on the elastic modulus (G’) (Fig. 2). It is believed, that at higher polymer concentrations particles are held in much closer proximity, as such are effected much more by particle deformation. The slower cooling rate allows the particles to form much more slowly, resulting in more ordered, stronger structures. Little effect is observed for the viscosity however, suggesting that particles interact with each other to a similar extent, with particles characterised on the microscale as they“squeeze” past each other.
  • NaCI (0.2 M) was prepared through the addition of dry crystals (1.16 g) to deionised water (100 ml) using a volumetric flask. The NaCI was then allowed to dissolve using an inverting technique to aid the process. Once fully dissolved the solutions were kept at ambient conditions until further use.
  • Gellan solutions were prepared by dissolving powdered polymer into water/NaCI solution so that the final concentrations post-processing were equal to 0.9% and 1.8% (w/v).
  • gellan powder was weighed out (4.5 g, 9.0 g) and added to 450 ml of deionised water. The mixture was allowed to heat to 95 °C under agitation, allowing the polymer to dissolve. Once fully dissolved, 25 ml of NaCI stock solution (0.2 M) was added to the solution resulting in a 10 mM concentration post-processing. The sol was then allowed to reach thermal equilibrium at 95 °C before processing. Processing of Gellan MS:
  • MS were prepared using a jacketed pin mill set to 20 °C.
  • Gellan sols were pumped using a peristaltic pump into the pin mill at 3 ml/min so that it entered the processing chamber at 40 °C.
  • water was pumped into the gellan stream (at a rate of 0.16 ml/min) so that they impinged, diluting the gellan sol to the final concentrations (0.9 and 1.8% (w/v), 10 mM NaCI).
  • the mixture was then cooled under shear (100, 500, 1000 and 2000 rmm) as it passed through the milling unit. On exiting, at 20 °C, the gel was packaged and stored at 4 °C until further testing.
  • a rheometer (TA, AR-G2) equipped with a sandblasted parallel plate (40 mm diameter, 1 mm gap height) was used to test all samples, at 20 °C.
  • Amplitude sweeps were obtained in strain controlled mode over a range of 0.1 to 100.0 %. Samples were loaded into the instrument and upper geometry lowered. Once trimmed, the sample was left to equilibrate at 20 °C prior to testing. Measurements were obtained at 1 Hz in a logarithmic fashion.
  • Viscosity profiles for the samples were obtained using a continuous ramp. Samples were loaded into the instrument and upper geometry lowered. Once trimmed, the sample was left to equilibrate at 20 °C prior to testing. Increasing shear was applied to the sample in rate controlled mode, between 0.1 and 600 s -1 over a 3-minute ramp, with data points obtained in a logarithmic fashion.
  • Tandon A Tovey JC, Sharma A, Gupta R, Mohan RR. Role of transforming growth factor Beta in corneal function, biology and pathology. Current molecular medicine. 2010;10(6):565-78.
  • IGF-IR IGF receptor I

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Abstract

La présente invention concerne des compositions d'hydrogel oculaires rhéofluidifiantes qui comprennent 0,1 à 5,0 % en poids (par exemple 0,1 à 3,5 % en poids ou 0,1 à 2,5 % en poids) d'un polymère formant des particules de microgel ; et 0,5 à 100 mM d'un sel d'ion métallique monovalent et/ou polyvalent en tant qu'agent de réticulation ; dispersés dans un véhicule aqueux. Les compositions d'hydrogel ont un pH dans la plage de 3 à 8 et la viscosité de la composition de gel diminue lorsque le gel est exposé à un cisaillement. Les compositions comprennent de la décorine. Les compositions peuvent également comprendre un antibiotique comme la gentamicine, et un anti-inflammatoire stéroïdien comme la prednisolone. Les compositions sont adaptées à un usage médical destiné au traitement de l'œil. Par exemple, les compositions sont adaptées à un usage destiné à la cicatrisation et/ou la prévention ou au traitement de la kératite microbienne.
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US17/311,561 US20220023206A1 (en) 2018-12-07 2019-12-09 Ocular hydrogel compositions
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WO2022006076A1 (fr) * 2020-07-01 2022-01-06 The Johns Hopkins University Compositions et méthodes de traitement oculaire

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